The present invention relates to a metabolite of all-trans-retinol, and more particularly to such a metabolite possessing a thiophene ring.
Retinol and its metabolites participate in many physiological processes, such as growth and development, reproduction, cellular differentiation and proliferation, and vision. Except in the visual system where the functional forms of a vitamin A have been well characterized (Wald, Science 162, 230-239, 1968), other biological functions of vitamin A are thought to be mediated by the action of retinoic acids (RAs) which interact with retinoic acid receptor (RAR) and retinoic acid X receptor (RXR) heterodimers or RXR homodimers and, therefore, regulate gene expression (Allenby et al, Proc. Natl. Acad. Sci. USA 90, 30-34, 1993; Nagpal et al, EMBO J. 12, 2349-2360, 1993). The discovery of RARs (Petkovich et al, Nature (London) 330, 444-450, 1987; Brand et al, Nature (London) 332, 850-353, 1988; Giguere et al, Nature (London) 330, 624-629, 1987; Benbrook et al, Nature, 333, 669-672, 1988; Krust et al, Proc. Natl. Acad. Sci. USA 86, 5310-5314, 1989; Zelent et al, Nature (London) 339, 714-717, 1989) and RXRs (Mangelsdorf et al, Nature (London) 345, 224-229, 1990; Mangelsdorf et al, Genes Dev. 6, 329-344 1992; Yu et al, Cell 67, 1251-1266, 1991) leads people to understand the mechanisms of RA action on the molecular level.
However, overwhelming data demonstrate that rats fed retinol free and all-trans-retinoic acid (atRA) supplemented diet fail to reproduce (Thompson et al, Proc. R. Soc. Lond B. Biol. Sci., 159, 510-535, 1964; Takahashi et al, J. Nutr. 105, 1299-1310, 1975; Wellik and DeLuca, Biol. Rep. 53, 1392-1397, 1995; Wellik et al, Am. J. Physiol. 292 (Endocrinol. Metab. 35), E25-E29, 1997). Although vitamin A-deficient, atRA supplemented (VAD-RAS) female rats can become pregnant after mating with healthy males, they invariably resorb their fetuses at approximately day 15 of gestation. Work by Wellik and DeLuca, Biol. Rep. 53, 1392-1397, 1995, showed that retinol is required no later than day 10 of gestation in order to prevent fatal resorption in the VAD-RAS female rats, and administration of a dose as little as 2 .mu.g on day 10 of gestation could prevent the resorption and allow parturition to complete.
Wellik and DeLuca, Arch. Biochem. Biophys. 330, 355-362, 1996 studied atROL metabolites in day 10 conceptuses of VAD-RAS rats. After administration of a dose of 2 .mu.g atROL into the 10-day pregnant female rats, an unknown metabolite was found in the conceptuses. The concentration of this metabolite increases through 6 hour post-dose. It did not react with phosphatase, glucuronidase or sulfatase, therefore, it was unlikely to be an excretion product. Further study showed that the metabolite was also found in several other tissues collected from the VAD-RAS female rats. Kidneys from the VAD-RAS female rats contained 70 times the amount of the metabolite found in the conceptuses. However, the small quantity of the metabolite produced in vivo was not sufficient for determination of the structure of the compound.
Based on the previous data by Wellik and DeLuca, Arch. Biochem. Biophys. 330, 355-362, 1996, the present study was focused on establishing an in vitro generation method which would obtain the unknown atROL metabolite in quantities required for its structural identification. The structure of this unknown metabolite was studied by ultraviolet (UV), infrared (FT-IR) and nuclear magnetic resonance (.sup.1 H NMR) spectroscopy as well as mass spectrometry.